Leveraging Programmable Integrases for Human Genome Engineering

利用可编程集成进行人类基因组工程

• 批准号: 10002492
• 负责人: Samuel Henry Sternberg
• 金额: $ 243万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-09 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10002492
• 关键词: Agriculture; Bacteria; Biological; Biology; Biomedical Research; Categories; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Double Strand Break; DNA Integration; DNA Repair; Disease; Elements; Engineering; Enhancers; Eukaryotic Cell; Event; Exhibits; Gene Expression; Genes; Genetic Diseases; Genetic Engineering; Genome; Genome engineering; Genomics; Guide RNA; Homologous Gene; Human Genome; Industry; Integrase; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Molecular; RNA; RNA library; Recombinant DNA; Research; Role; Site; Specificity; System; Technology; Transposase; Untranslated RNA; Virus; Vision; Work; engineered nucleases; genetic payload; homologous recombination; human disease; human model; improved; insight; molecular array; nuclease; programs; site-specific integration; tool

PROJECT SUMMARY LEVERAGING PROGRAMMABLE INTEGRASES FOR HUMAN GENOME ENGINEERING The genetic engineering toolbox comprises a diverse array of molecular machineries for genome manipulation, and DNA insertion methods have arguably had the largest impact on biomedical research. Gene knock-ins are used in the clinic to treat genetic diseases and cancer, in industry to manufacture biologics, in agriculture to improve crops, and in research to generate models of human disease, among many other uses. These applications generally depend on either random integration mediated by viruses and transposases, or site- specific integration mediated by homologous recombination and gene editing. The former category exhibits high efficiency but little specificity, whereas the latter category is inherently precise but reliant on cellular factors and thus ineffective. Only recently has a new molecular functionality been discovered that is both fully autonomous and also highly accurate: programmable integrases directed by CRISPR RNAs. CRISPR systems have revolutionized biology over the past decade because of how easily one can program CRISPR-associated nucleases with guide RNAs to introduce DNA double-strand breaks, the precursor to DNA repair. Whereas the inability to easily redesign engineered nucleases previously stalled gene-editing technology, the discovery of RNA-guided DNA targeting eliminated this critical bottleneck. A similar bottleneck for engineered integrases is now ready for elimination. My central vision is to develop programmable, RNA-guided integrases as a powerful new platform technology for human genome engineering. Building on our recent work that deciphered sequence determinants of this technology in bacteria, as well as parallel studies that expanded the CRISPR–Cas subtypes that function robustly in mammalian cells, we will embark upon a systematic effort to build the capabilities for employing these multi-subunit integrases in eukaryotic cells. We will then develop the first tools for performing simultaneous, multiplexed DNA insertion events across thousands of distinct genomic target sites using guide RNA libraries. This approach will enable us to probe fundamental questions regarding the role of noncoding elements such as enhancers and insulators in regulating gene expression. Furthermore, we will harness orthogonal integrases to execute highly programmed translocation events and study the role of complex genome rearrangements in disease and cancer. Our studies will contribute powerful new tools to the genetic engineering toolbox and open the door to genomic manipulations that are inaccessible with any other experimental approach.

项目概要 利用可编程集成进行人类基因组工程 基因工程工具箱包括用于基因组操作的多种分子机器， DNA插入方法存在争议，对生物医学研究影响最大的是基因敲入。 在临床中用于治疗遗传疾病和癌症，在工业中用于制造生物制剂，在农业中用于 改良作物、研究生成人类疾病模型以及许多其他用途。 应用通常取决于病毒和转座酶介导的随机整合，或位点- 由同源重组和基因编辑介导的特异性整合，前一类表现出高水平。 效率但特异性很小，而后一类本质上是精确的，但依赖于细胞因素和 直到最近才发现一种新的分子功能，它是完全自主的。 而且高度准确：由 CRISPR RNA 指导的可编程整合酶。 CRISPR 系统在过去十年中彻底改变了生物学，因为人们可以轻松地进行编程 CRISPR 相关核酸酶与引导 RNA 可引入 DNA 双链断裂（DNA 的前体） 修复，而无法轻松地重新设计工程核酸酶先前阻碍了基因编辑技术， RNA 引导的 DNA 靶向的发现消除了工程设计的一个类似瓶颈。 整合酶现在已准备好被淘汰。 我的中心愿景是开发可编程的、RNA 引导的整合作为一个强大的新平台 人类基因组工程技术以我们最近破译序列决定因素的工作为基础。 这项技术在细菌中的应用，以及扩展 CRISPR-Cas 功能亚型的平行研究 在哺乳动物细胞中，我们将开展系统性的努力，以建立利用这些技术的能力 然后，我们将开发第一个用于同时执行的工具。 使用向导 RNA 文库在数千个不同的基因组目标位点上进行多重 DNA 插入事件。 这种方法将使我们能够探讨有关非编码元素作用的基本问题，例如 此外，我们将利用正交整合酶来调节基因表达。 执行高度编程的易位事件并研究复杂基因组重排的作用 我们的研究将为基因工程工具箱提供强大的新工具并开放。 这是任何其他实验方法都无法实现的基因组操作的大门。